Arch Virol (2000) 145: 1947–1961 Mapping of functional domains on the influenza A virus RNA polymerase PB2 molecule using monoclonal antibodies M. Hatta 1 , Y. Asano 2,3 , K. Masunaga 4,5 , T. Ito 6 , K. Okazaki 1 , T. Toyoda 4 , Y. Kawaoka 7 , A. Ishihama 2 , and H. Kida 1 1 Department of Disease Control, Graduate School of Veterinary Medicine, Hokkaido University, Sapporo, Japan 2 Department of Molecular Genetics, National Institute of Genetics, Mishima, Shizuoka, Japan 3 Rational Drug Design Laboratories, Fukushima, Fukushima, Japan 4 Department of Virology, Kurume University School of Medicine, Kurume, Fukuoka, Japan 5 Department of Pediatrics, Kurume University School of Medicine, Kurume, Fukuoka, Japan 6 Department of Veterinary Public Health, Faculty of Agriculture, Tottori University, Tottori, Japan 7 Department of Pathobiological Sciences, School of Veterinary Medicine, University of Wisconsin-Madison, Madison, Wisconsin, U.S.A. Accepted January 5, 2000 Summary. Monoclonal antibodies against the PB2 of A/Puerto Rico/8/34 (A/PR/ 8/34) (H1N1) were prepared in order to define the functional domains of the RNA polymerase of influenza virus. The fifteen monoclonal antibodies that were generated were divided into 4 groups on the basis of ELISA binding to PB2 or its peptide fragments. Six Group I antibodies that bound to the PB2 N-terminal region (amino acids 1–104) did not inhibit transcription by the viral ribonucleoprotein complex. A single Group II antibody recognizing the region of amino acids 206– 259 inhibited ApG-primed transcription. Groups III and IV antibodies bound to the C-terminal region of amino acids 660–759. Of these, Group III antibodies inhibited transcription. The present results identify multiple monoclonal antibody binding domains in PB2, two of which, when bound by antibodies, negatively affect viral RNA transcription. Introduction Influenza A virus contains 8 segments of linear, negative-sense, single stranded RNA. In virus particles, the genomic RNAs (vRNAs) are associated with the RNA-